Reconstruction of craniofacial skeletal defects from both congenital and acquired etiologies can often present a daunting challenge. Contemporary strategies employ bone grafting and free tissue transfer, but donor site morbidity, as well as suboptimal restoration of form and function, continues to drive the development of novel approaches. The field of regenerative medicine holds significant promise to address this need, employing cellular-based strategies to replace damaged or deficient tissues. Despite tremendous advances, however, several complex issues remain. Clinical application of stem cells often involve placement into hostile wounds that are hypoxic and possess dramatically upregulated inflammatory mediators. Within such a harsh environment, cellular survival is uncertain, and the utility of implanted cells can be severely compromised. In light of this, there s growing recognition that simple placement of stem cells into a wound is not sufficient to ensure maximal tissue regeneration. While investigators have focused on promoting angiogenesis or limiting inflammation, a novel approach would be to enhance pro-survival pathways within the transplanted cells themselves through upregulation of Bcl-2, a regulator of apoptosis, for bone regeneration. Importantly, increased expression of Bcl-2 has been shown to reduce apoptosis without limiting subsequent differentiation capacity in mesenchymal cells. However, limited expression of Bcl-2 is necessary to avoid any tumorigenic risk. We will therefore employ a non-integrating, non-viral minicircle vector to transfect human adipose-derived stromal cells and evaluate their subsequent viability and bone forming capacity. Furthermore, to maximize the translational potential of our approach, we will develop an innovative scaffold incorporating polyethylenimine- encapsulated superparamagnetic iron oxide nanoparticles complexed to our Bcl-2 expressing minicircle. This will allow for an off the shelf approach where freshly harvested cells can be directly seeded onto scaffolds and placed back into the patient for in situ transfection. We will evaluate the ability for this novel strategy to enhance survival and bone regenerative ability of human adipose-derived stromal cells implanted into non- healing critical-sized calvarial defects in immunocompromised mice. Collectively, the experiments proposed will determine the ability for Bcl-2 upregulation to promote bone repair and facilitate development of new clinical applications to repair large skeletal defects.